Infectious diseases including respiratory infections continue to be a major health concern. In many cases infections establish when the innate mucosal defense fails. Antimicrobial peptides have been recognized as important contributors to the first line of defense. Our recent work has provided evidence that host-derived lipids represent a new class of antimicrobial effector molecules that are integral to the inherent antimicrobial activity of mucosal fluids exerting bactericidal activity alone and in synergism with antimicrobial peptides. We have further identified, for the first time, cholesteryl esters (CE's) as antimicrobial effector molecules. However, there is a major gap in knowledge with respect to the regulation of antimicrobial lipids, their mode of action, and their interaction with antimicrobial peptides. The research proposed here will answer the important questions how antimicrobial CE's are regulated in the context of infection and inflammation in the respiratory tract, which of the CE's exert most potent antibacterial activity, and how they interface with antimicrobial peptides. To identify key regulators of epithelial CE production and the most potent antibacterial CE's we will use an infection model with polarized airway epithelial cells and opportunistic respiratory tract pathogens that cause infection when the mucosal barrier function is impaired. We will assess the expression of key enzymes involved in CE biosynthesis and quantify antibacterial CE's in response to ligands for pattern recognition receptors such as bacterial cell wall products, and pro-inflammatory cytokines that act on epithelial cells, for example IL1b, IL17A. We will employ quantitative RT-PCR, biochemical analysis, and antibacterial assays. We will validate our results with primary epithelial cells established from sino-nasal mucosa obtained as surgical excess material, and confirm key regulators with siRNA methodology. We will dissect the relative contributions of antimicrobial lipids and antimicrobial peptides to the mucosal innate defense employing antibacterial assays, and bacterial challenge experiments with epithelial cells and selective inhibition of effector molecule production with siRNA technology. Upon completion of the proposed work we will be in the position to study the mode of action of antimicrobial lipids in vitro and moving into an animal model. Our study is innovative and highly significant. The lipid-arm of innate defense is an emerging concept and this research promises a high impact on public health in multiple ways. A better understanding of how to manipulate the natural production and secretion of antimicrobial lipids, in particular CE's, can lead to novel immunotherapies for infectious diseases caused by a deficiency in lipid production. The identification of antibacterial CE's and synergistically acting antimicrobial peptides may guide the development of novel lipid-antibiotics. Last, not least, this research may lead to the discovery of novel virulence factors of pathogenic microbes that circumvent host defenses mediated by antimicrobial lipids in the respiratory tract and on other mucosal surfaces. PUBLIC HEALTH RELEVANCE: Host-derived fats have emerged as important factors in the natural defense of our body surfaces. This proposal seeks to elucidate the regulation and spectrum of activity of these novel anti-infectious agents and their interplay with well established key players in the natural defense system, namely natural protein antibiotics. This research may lead to novel anti-infectious therapy and prophylaxis and may give new insight into host- pathogen interaction, which is highly relevant considering the burden infectious diseases pose on public health.